Novel Methylated DNA Markers in the Surveillance of Colorectal Cancer Recurrence

Abstract

Purpose: We aimed to assess the concordance of colorectal cancer-associated methylated DNA markers (MDM) in primary and metastatic colorectal cancer for feasibility in detection of distantly recurrent/metastatic colorectal cancer in plasma.

Experimental design: A panel of previously discovered colorectal cancer-associated MDMs was selected. MDMs from primary and paired metastatic colorectal cancer tissue were assayed with quantitative methylation-specific PCR. Plasma MDMs were measured blindly by target enrichment long-probe quantitative-amplified signal assays. Random forest modeling was used to derive a prediction algorithm of MDMs in archival plasma samples from primary colorectal cancer cases. This algorithm was validated in prospectively collected plasma samples from recurrent colorectal cancer cases. The accuracy of the algorithm was summarized as sensitivity, specificity, and area under the curve (AUC).

Results: Of the 14 selected MDMs, the concordance between primary and metastatic tissue was considered moderate or higher for 12 MDMs (86%). At a preset specificity of 95% (91%-98%), a panel of 13 MDMs, in plasma from 97 colorectal cancer cases and 200 controls, detected stage IV colorectal cancer with 100% (80%-100%) sensitivity and all stages of colorectal cancer with an AUC of 0.91 (0.87-0.95), significantly higher than carcinoembryonic antigen [AUC, 0.72 (0.65-0.79)]. This panel, in plasma from 40 cases and 60 healthy controls, detected recurrent/metastatic colorectal cancer with 90% (76%-97%) sensitivity, 90% (79%-96%) specificity, and an AUC of 0.96 (0.92-1.00). The panel was positive in 0.30 (0.19-0.43) of 60 patients with no evidence of disease in post-operative patients with colorectal cancer.

Conclusions: Plasma assay of novel colorectal cancer-associated MDMs can reliably detect both primary colorectal cancer and distantly recurrent colorectal cancer with promising accuracy.

Figure 1:

(A) Study rationale for using methylated DNA markers compared to mutation-based biomarker in metastatic colorectal cancer and B) overview of three parallel studies to address primary aims. 1) Primary and Metastatic Tissue MDM Concordance Study to assess concordance of MDMs between primary and metastatic CRC over time; 2) MDM Plasma CRC Early Primary Detection Study to measure sensitivity and specificity of MDMs for early detection of primary CRC; 3) MDM Plasma CRC Surveillance Study to measure sensitivity and specificity of MDMs for detection of recurrent and metastatic CRC. MDM: methylated DNA marker; CRC: colorectal cancer; NED: no radiographic evidence of disease.

Figure 2:

Differences in tissue methylated DNA marker (MDM) levels between paired primary and metastatic colorectal cancer (CRC). Differences expressed as median and IQR fold change when dividing primary tumor level by paired metastatic tumor level. (A) Overall, (B) between primary and synchronous metastatic CRC; (C) between primary and metachronous metastatic CRC; (D) between left-side primary and metastatic CRC; and (E) between right-side primary and metastatic CRC. Fold change values >1 indicate higher MDM levels in the primary; values <1 indicate higher MDM levels in the metastasis.

Figure 3:

The performance of individual MDMs, CEA, and a panel of MDMs in plasma to distinguish early CRC from healthy controls in Early Primary CRC Detection Pilot Study using MDMs in Plasma. MDM: methylated DNA marker; CEA: carcinoembryonic antigen; CRC: colorectal cancer; AUC: area under the curve.

Figure 4:

Performance of the cross-validated model from Early Primary CRC Detection Pilot Study using MDMs in Plasma in patients from MDM Plasma CRC Surveillance Study. (A) The distribution of the probability of CRC being present in the surveillance dataset for each of the patient groups. The blue shaded area indicates where the algorithm derived from training data made positive calls in the test set. (B) ROC curves of MDM models with and without CEA and CEA alone for recurrent CRC versus normal controls with corresponding AUCs and 95% confidence intervals. CRC: colorectal cancer; ROC: receiver operating characteristic; NED: no radiographic evidence of disease; AUC: area under the curve; MDM: methylated DNA marker.